Hantaviruses are RNA viruses that cause hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). HPS and HFRS are characterized by fever, myalgia, rapid onset of a vascular leak syndrome, hemoconcentration, and thrombocytopenia. In HPS, the lung is the prominent target organ; while, in HFRS, the kidney is the prominent target organ. Hantaviruses are NIAID category A priority pathogens with regards to biodefense, as they can produce severe, potentially fatal, diseases, are transmitted by aerosol, and do not have effective vaccines or specific therapeutics. The goal of this project is to understand the immunologic mechanisms that lead to HFRS. Several lines of evidence suggest that HFRS is not caused by direct cytopathic effects of hantaviruses, but rather by exuberant host immunopathological responses. This project will rely on samples provided from a prospective cohort study of Puumala (PUU) virus infections, a HFRS-associated hantavirus in Finland. The first aim will be to characterize dendritic cell functions and humoral immune responses that affect the PUU virus burden, using flow cytometry, antibody detection assays, and quantitative viral RT-PCR. The second aim will be to analyze the patterns and temporal regulation of cellular immune responses throughout acute PUU virus infection. ELISAs, multiplex immunoassays, quantitative RT-PCR, and genomic screening techniques will be used to examine immune response mediators in a comprehensive fashion, along with virus levels and disease severity. The third aim will be to characterize the antigen specificity and behavior of T lymphocyte responses during and after PUU virus infection. CD8+ and CD4+ T cell epitopes from PUU virus proteins will be identified using cell cloning techniques, ELISPOTs, cytotoxic T lymphocyte (CTL) assays, and mapping with overlapping synthetic peptides. Effector mechanisms of vascular leakage will be studied by examining interactions between endothelial cells and PUU virus-specific T cell clones. Peptide stimulation with intracellular cytokine staining and peptide-HLA Class I and II tetramers will be used to identify and quantify antigen-specific T cell responses across a spectrum of PUU virus disease. Elucidation of the immunopathogenetic mechanisms in PUU virus infection will contribute to the development of effective vaccine strategies and immune-based therapies of HFRS and HPS.